Project Summary/Abstract This proposal seeks to reveal the underlying structural mechanism of consensus hyperactivation of the nuclear receptor (NR) ROR? driving maximal transcriptional output of target genes. To achieve this goal, I will perform detailed mechanism of action studies on a range of novel synthetic agonists of ROR? to further our understanding the molecular mechanism driving their ?functional selectivity? and to evaluate the physiological role of ROR?. I hypothesize that ligand-dependent structural perturbations manipulate coregulator interactions and PTM status of the receptor to directly influence gene program regulation. I aim to correlate structure based measurements and proteogenomic studies with biochemical and biological activity of synthetic agonists for ROR? and contrasting this to results obtained using repressive antagonist/inverse agonist ligands. ROR? agonists have already been developed with various potencies, binding modes, and receptor activation potential in cell based assays7,8. To accomplish the goals of my research project, I propose the following specific aims. Aim 1: Develop a comprehensive structural model for ligand activation of ROR?. I will perform structure determination of agonist-LBD complexes via X-ray co-crystallography and will determine ligand-dependent perturbation in receptor plasticity with hydrogen/deuterium exchange mass spectrometry (HDX-MS). As a proxy for receptor conformation, a coregulator interaction screen will be developed to determine ligand effects on specificity and affinity for coregulator recruitment. Crosslinking coupled with mass spectrometry (XL-MS) and HDX-MS will be conducted to quantitate ligand-dependent effects on LBD conformation ensembles and coregulator interactions as well as determining specific NR box motifs receptor interactions. These comprehensive biophysical measurements will be used to cluster compounds with similar properties where the functional consequences of these structural perturbations will be determined in Aim 2. Aim 2: Validation of structural model in cultured cells in vitro and ex vivo for ROR? agonists. Agonist activity in cultured cells will be characterized in relevant systems by expression profiling of ROR? target genes and coregulator associations using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). The murine T lymphoma EL4 cell line will be used for initial compound screening by monitoring transcript modulation. This system will also be used to develop RIME methodology. Induced T helper 17 cells (iTH17) will serve as a more physiologically relevant system for agonist characterization. iTh17 target gene programs and coregulator perturbations will be determined by RNA-seq and RIME, respectively. Co-immunoprecipitation with western blotting and chromatin immunoprecipitation experiments will be conducted to confirm findings from RIME.